Novel compositions and methods for producing recombinant aav

ABSTRACT

Provided herein are nucleic acid constructs, host insect cells, and methods for producing recombinant AAV capsids with high potency at high yield.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. ProvisionalApplication 62/943,715, filed Dec. 4, 2019, the content of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted electronically in ASCII format. The Sequence Listing is herebyincorporated by reference in its entirety. The ASCII copy, created onNov. 30, 2020, is named 025297_WO015_SL.txt and is 111,015 bytes insize.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV) is a small non-enveloped virus belonging tothe family Parvoviridae and the genus Dependoparvovirus. AAV is composedof a single-stranded DNA genome packaged into capsids assembled fromthree capsid proteins—viral protein (VP) 1, VP2, and VP3—at anapproximate molar ratio of 1:1:10 (Berns and Parrish (2007) Parvoviridaein Fields Virology (Knipe and Howley, eds., 5th ed. Philadelphia:Wolters Kluwer Health/Lippincott Williams & Wilkins); Wang et al., NatRev Drug Discov. (2019) 18 (5):358-78). The capsid proteins are encodedby a single capsid (cap) gene and are generated by alternative splicingand differential codon usage (id.). VP1 is the largest capsid protein(81.6 kD). VP2 (66.6 kD) is an N-terminal truncated form of VP1. VP3(59.9 kD) is an N-terminal truncated form of VP2. See, e.g., Cecchini etal., Hum Gene Ther. (2011) 22:1021-30.

Recombinant AAV (rAAV) has been intensively explored as a vector forgene therapy and DNA vaccines in humans. For rAAV production inmammalian cells, a helper virus (e.g., adenovirus, vaccinia, orherpesvirus) is needed. Over the years, several modifications have beenmade to facilitate the production of rAAV, including (1) identifying andcloning the minimal set of helper proteins (adenovirus E1, E2A, E4, andVA), (2) providing the AAV Rep (Rep78, Rep68, Rep52, and Rep40) andcapsid proteins in trans, and (3) developing a transgene system thatallows packaging of DNA sequences flanked by the AAV Inverted TerminalRepeats (ITRs) (Samulski et al., J Virol. (1989) 63 (9):3822-8). Whenthese three components are introduced into mammalian cells, rAAVcontaining a transgene of interest can be readily purified. RecombinantAAV so produced has been used in clinical trials (Clement and Grieger,Mol Ther Methods Clin Dev. (2016) 3:16002).

The need for scaled-up rAAV production for larger clinical trials andcommercialization has led to the development of insect cell-basedproduction systems that utilize baculoviral vectors to express the Repand capsid proteins and to carry the coding sequence for atransgene-containing AAV vector genome, which is packaged into rAAVcapsids. Insect cell-based manufacturing of recombinant AAV (rAAV)offers several advantages over mammalian cell-based rAAV manufacturing,including scalability of non-adherent cells and cost savings due to theuse of serum-free growth conditions. Such systems also do not requireadenovirus helper functions (see, e.g., Urabe et al., Hum Gene Ther.(2002) 13:1935-43; Urabe et al., J Vir. (2006) 80 (4):1874-85; Chen etal., Mol Ther. (2008) 16 (5):924-30; Smith et al., Mol Ther. (2009) 17(11):1888-96; and Mietzsch et al., Hum Gene Ther Methods (2017) 28(1):15-22). While the baculovirus-insect cell system has beensuccessfully utilized for rAAV production at multiple scales, it hasbeen observed that rAAV generated in insect cells has reduced VP1content (e.g., with a VP1:VP2:VP3 ratio of approximately 1:1:30 to1:1:60) and consequently reduced potency, as compared to rAAV producedin mammalian cells (see, e.g., Urabe et al., 2002, supra; Kohlbrenner etal., Mol Ther. (2005) 12 (6):1217-25; Urabe et al., 2006, supra;Aslanidi et al., Proc Natl Acad Sci USA (2009) 106 (13):5059-64;Kondratov et al., Mol Ther. (2017) 25 (12):2661-75; and Mietzsch et al.,supra). Several efforts have been made to improve the capsid ratios bymodifying the start codon context of VP1 (see, e.g., Kondratov et al.,supra; Mietzsch et al., supra).

Thus, there remains a need for an improved baculovirus-insect cellsystem for producing rAAV at industrial scales.

SUMMARY OF THE INVENTION

The present disclosure provides baculovirus-insect cell systems forproducing potent recombinant AAV at high yield. In one aspect, thepresent disclosure provides an insect cell (e.g., an Sf9 or Sf21 cell)comprising a first baculoviral vector comprising an expression cassettefor Rep78 and a second baculoviral vector comprising an expressioncassette for Rep52, wherein the first vector and the second vectorfurther comprise (i) an expression cassette for VP1 and an expressioncassette for VP2/VP3, respectively; or (ii) an expression cassette forVP2/VP3 and an expression cassette for VP1, respectively. In someembodiments, one or both of the first and second vectors are stablyintegrated into the genome of the insect cell.

In some embodiments, the Rep78 expression cassette and the Rep52expression cassette comprise identical insect promoters. In someembodiments, the Rep78 expression cassette comprises a non-canonicalstart codon for the Rep78 coding sequence, wherein the codon isoptionally ACG, TTG, GTG, or CTG.

In some embodiments, the VP1 expression cassette and the VP2/VP3expression cassette comprise identical insect promoters. In someembodiments, the VP1, VP2, and VP3 proteins comprise amino acidsequences from the same AAV serotype, or from more than one AAVserotype. In certain embodiments, the VP1, VP2, and/or VP3 proteinscomprise amino acid sequences from AAV1, AAV2, AAV3 (e.g., AAV3B), AAV6,and/or AAV9. In certain embodiments, the Rep78 and Rep52 proteins arederived from a different AAV serotype from the VP1, VP2, and/or VP3proteins.

In some embodiments, the Rep78, Rep52, VP1, and VP2/VP3 expressioncassettes each comprise an insect promoter selected from a polyhedronpromoter, an IE-1 promoter, and a p10 promoter.

The insect cell herein may further comprise a coding sequence for arecombinant AAV genome, wherein the recombinant AAV genome comprises anexpression cassette for a transgene of interest that is under thetranscriptional control of a mammalian promoter, and an AAV invertedterminal repeat (ITR) on both termini. In some embodiments, the codingsequence for the recombinant AAV genome is located on the first orsecond vector, or is located on a third vector. The transgene ofinterest may encode, for example, a therapeutic protein, including,without limitation: a protein whose function is lacking or deficient ina genetic disease (e.g., a lysosomal storage disease, or a hemophilia),such as an enzyme (for use in an enzyme replacement therapy) and a bloodclotting factor (for use as replacement factor); and a protein forregulating gene expression (e.g., a zinc finger protein (ZFP)transcription factor). The transgene of interest may also encode a geneediting protein, such as a zinc finger nuclease (ZFN), a ZFP deaminase,a ZFP recombinase, a TALEN, a CRISPR Cas protein, and a CRISPR Cpfprotein. The transgene may also encode an interfering RNA molecule suchas a small hairpin RNA.

In some embodiments, the VP1 protein and/or the VP2 protein comprise twoor more mutations at residues 157, 162, 164, 179, 188, 194, 196, 197,200, and 201 (numbering according to SEQ ID NO:1) relative to wildtypeVP1 and VP2 proteins, respectively. As used herein, “numbering accordingto SEQ ID NO:1” means amino acid residue positions in SEQ ID NO:1, orthe corresponding amino acid residue positions in a different VP1sequence (e.g., VP1 sequence from a serotype other than AAV6). In someembodiments, the VP1 protein and/or the VP2 protein comprise two or moremutations selected from the group consisting of S157A, T162S, Q164A,S179T, L188I, T194A, A196S, A197G, P200S, and T201L (numbering accordingto SEQ ID NO:1). In further embodiments, the VP1 protein and/or the VP2protein comprise all of these ten mutations.

In some embodiments, the VP1 protein further comprises one or moremutations at residues 67, 81, 84, 85, and 92 relative to wildtype VP1protein (numbering according to SEQ ID NO:1). In certain embodiments,the VP1 protein comprises one or more mutations selected from the groupconsisting of A67E, Q81R, K84D, A85S, and R92K. In further embodiments,the VP1 protein comprises all of these five mutations.

In some embodiments, the VP1 protein and the VP2 protein disclosedherein are identical to the VP1 protein and the VP2 protein,respectively, of AAV6 but for the mutations. In particular embodiments,the VP1 protein and/or the VP2 protein are derived from AAV6 andcomprise mutations S157A, T162S, Q164A, S179T, L188I, T194A, A196S,A197G, P200S, and T201L relative to wildtype AAV6 VP1 and VP2 proteins,respectively.

In some embodiments, the VP1-expressing vector also comprises anexpression cassette for assembly-activating protein (AAP), wherein theAAP comprises one or more mutations at residues 8, 10, 12, 17, 21, and22 (numbering according to SEQ ID NO:10) relative to wildtype AAPprotein. As used herein, “numbering according to SEQ ID NO:10” meansamino acid residue positions in SEQ ID NO:10, or the corresponding aminoacid residue positions in a different AAP sequence (e.g., AAP sequencefrom a serotype other than AAV6). In certain embodiments, the AAPcomprises one or more (e.g., all) mutations selected from the groupconsisting of P8Q, H10L, L12Q, Q17P, L21Q, and L22V. In particularembodiments, the AAP is identical to wildtype AAP of AAV6 but for theAAP mutations.

In particular embodiments, the VP1 expression cassette comprises acoding sequence for SEQ ID NO:7, with or without the first amino acid(e.g., nucleotides 18-151 of SEQ ID NO:14). In further embodiments, thecapsid expression cassettes comprise 10 or more (e.g., 20 or more, 30 ormore, 40 or more, 50 or more, 75 or more, 100 or more, 125 or more, or150 or more) contiguous nucleotides, or the entire nucleotide sequence,of SEQ ID NO:14.

In some embodiments, the VP1 protein comprises one or more mutations atresidues 81, 84, 85, and 92 relative to wildtype VP1 protein (numberingaccording to SEQ ID NO:1). In some embodiments, the VP1 proteincomprises one or more mutations selected from the group consisting ofQ81R, K84D, A85S, and R92K. In further embodiments, the VP1 proteincomprises all of these four mutations. In particular embodiments, theVP1 protein further comprises a mutation at residue 67, e.g., an A67Emutation. In some embodiments, the VP1 protein is identical to the VP1protein of AAV6 but for the mutation(s). In certain embodiments, the VP1protein comprises mutations A67E, Q81R, K84D, A85S, and R92K relative towildtype AAV6 VP1.

In particular embodiments of the present insect cell AAV productionsystems, the VP1 comprises SEQ ID NO:1, 7, or 16 with or without thefirst amino acid residue; the VP2 comprises amino acid residues 138-736or 139-736 of SEQ ID NO:1 or 7, or comprises SEQ ID NO:18 with orwithout the first amino acid residue; the VP3 comprises amino acidresidues of 204-736 or 205-736 of SEQ ID NO:1 or amino acid residues203-736 or 204-736 of SEQ ID NO:7, or comprises SEQ ID NO:19 with orwithout the first amino acid residue.

In some embodiments, the VP1 comprises SEQ ID NO:24 with or without thefirst amino acid residue; the VP2 comprises amino acid residues 138-736or 139-736 of SEQ ID NO:24; and the VP3 comprises amino acid residues of203-736 or 204-736 of SEQ ID NO:24.

In some embodiments, the VP1 comprises SEQ ID NO:25 with or without thefirst amino acid residue; the VP2 comprises amino acid residues 138-735or 139-735 of SEQ ID NO:25; and the VP3 comprises 203-735 or 204-735 ofSEQ ID NO:25.

In some embodiments, the VP1 comprises SEQ ID NO:26 with or without thefirst amino acid residue; the VP2 comprises amino acid residues 138-736or 139-736 of SEQ ID NO:26; and the VP3 comprises amino acid residues of203-736 or 204-736 of SEQ ID NO:26.

In some embodiments, the VP1 comprises SEQ ID NO:27 with or without thefirst amino acid residue; the VP2 comprises amino acid residues 138-736or 139-736 of SEQ ID NO:27; and the VP3 comprises amino acid residues of203-736 or 204-736 of SEQ ID NO:27.

In some embodiments, the Rep78 comprises SEQ ID NO:21 with or withoutthe first amino acid residue; and/or the Rep52 comprises SEQ ID NO:23with or without the first amino acid residue.

These embodiments encompass helper proteins (Rep78, Rep52, VP1, VP2, andVP3) that do not contain the first amino acid residue encoded by thecoding sequence (i.e., the amino acid encoded by the start codon)because the producing cell cleaves off that first amino acid residue.These embodiments also encompass helper proteins whose first amino acidresidues are encoded by a non-canonical start codon such as thosedescribed herein or otherwise known in the art.

In another aspect, the present disclosure also provides a recombinantAAV virion produced in the insect cell herein, its use in treating ahuman patient in need of the transgene, its use in the manufacture of amedicament for such treatment, and a pharmaceutical compositioncomprising the recombinant AAV virion and pharmaceutically acceptablecarrier.

In another aspect, the present disclosure provides a method of producinga recombinant AAV virion, comprising providing an insect cell herein,culturing the insect cell under conditions that allow expression of therecombinant AAV genome and packaging of the recombinant AAV genomewithin an AAV capsid comprising the VP1, VP2, and VP3 proteins, andisolating the recombinant AAV from the culture. Also provided are acombination (e.g., a composition) of the expression cassettes for makingthe AAV virion in insect cells.

Other features, objectives, and advantages of the invention are apparentin the detailed description that follows. It should be understood,however, that the detailed description, while indicating embodiments andaspects of the invention, is given by way of illustration only, notlimitation. Various changes and modification within the scope of theinvention will become apparent to those skilled in the art from thedetailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the alignment of the VP1/VP2 amino acid sequences from AAVserotypes 3B, 5, 6, 8, and 9 between amino acid residues 155 and 207(numbering based on consensus sequence). N-terminal Edman degradationsequencing was performed on the proteolytic cleavage products cored froman SDS-PAGE gel to identify the proteolytic cleavage site in AAV6VP1/VP2. The cleavage site maps to residues G₁₉₀E₁₉₁. Multiple sequencealignment was generated using the Geneious software package and defaultsettings for ClustalW alignment. The VP3 start site is indicated by thetriangle at residue 205. FIG. 1A discloses SEQ ID NOs:28-33,respectively, in order of appearance.

FIG. 1B shows the alignment of the VP1/VP2 amino acid sequences from AAVserotypes 1, 2, 3B, 4, and 6-11 between amino acid residues 155 and 205(numbering based on consensus sequence). Multiple sequence alignment wasgenerated using the Geneious software package and default settings forClustalW alignment. FIG. 1B discloses SEQ ID NOs:34-38, 73, and 39-43,respectively, in order of appearance.

FIG. 1C is a table comparing corresponding amino acid positions from AAVserotypes 1, 4, 6, 7, 8, and 11 between amino acid residues 158-203 asaligned in FIG. 1B. The table indicates amino acid mutations that mayprevent proteolytic cleavage in these serotypes. Amino acid residuenumbers are based on the consensus sequence shown in FIG. 1B.

FIG. 2 shows the substitution of a 45-amino acid stretch of an AAV9VP1/VP2 (SEQ ID NO:4) for the corresponding region in the AAV6 VP1/VP2sequence (SEQ ID NO:3), which abolishes proteolytic cleavage of theresultant chimeric VP1/VP2 proteins (“AAV6/9 VP1/VP2”). The figure showsthe alignment of the transplanted AAV9 Cap nucleotide sequence and theAAV6 Cap nucleotide sequence that is replaced in AAV9 Transplant; thearrow “A” indicates the AAP start codon. AAV9 Transplant: baculoviralhelper vector whose AAV6 Cap gene contains a sequence from the AAV9 Capgene. FIG. 2 discloses SEQ ID NOs:44-45, respectively, in order ofappearance.

FIGS. 3A and 3B illustrate variants of AAV6/9 VP1/VP2 preventedcleavage. FIG. 3A: a sequence alignment of Variants 1, 2, 3, and 4 ofAAV6/9 VP1/VP2 (numbering according to SEQ ID NO:1). FIG. 3B: partialalignment of the cap/AAP nucleotide sequences and the AAP amino acidsequences (in rectangle) of AAV6, AAV9 Transplant, Variant 1, Variant 2,Variant 3, and Variant 4. The AAP amino acid sequence of AAV9 Transplanthas six mutations (P8Q, H10L, L12Q, Q17P, L21Q, and L22V) relative tothat of AAP6 in the indicated region, while the AAP amino acid sequencesof Variants 1-3 each have only two mutations (L21Q and L22V). Variant 4has the native AAP6 sequence in the indicated region. FIG. 3A disclosesSEQ ID NOs:46-51 and FIG. 3B discloses SEQ ID NOs:52, 67, 53, 68, 54,69, 55, 70, 56, 71, 57, and 72, all respectively, in order ofappearance.

FIG. 4A shows the alignment of the assembly-activating protein (AAP)amino acid sequences from AAV serotypes 1, 2, 3B, 4, and 6-11 in theindicated region. Amino acid residue numbers are based on the consensussequence. Multiple sequence alignment was generated using the Geneioussoftware package and default settings for ClustalW alignment. FIG. 4Adiscloses SEQ ID NOs:58-62, 8, 63-64, 9, and 65-66, respectively, inorder of appearance.

FIG. 4B shows the amino acid differences in the indicated AAP region(consensus numbering) between (i) AAV1, AAV2, AAV4, AAV6, AAV7, AAV8,AAV10, or AAV11 and (ii) AAV9 or AAV3B. Amino acid residue numbers arebased on the consensus sequence shown in FIG. 4A.

FIG. 5 is a schematic diagram showing two baculoviral helper systems forproducing AAV in insect cells. The start codons for the Rep78, Rep52,VP1, and VP2/VP3 coding sequences, along with their transcriptiondirection, are shown. Both systems involve two baculoviral vectors. Acoding sequence for a recombinant AAV genome comprising a gene ofinterest (GOI) flanked by a pair of AAV inverted terminal repeats (ITR)can be placed on a separate baculoviral vector, or placed on the samevector as the Rep52 coding sequence.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides improved baculovirus-insect cell systemsand related compositions for producing potent rAAV capsids at highyield. The present rAAV production systems utilize dual baculoviralhelper vectors. The first vector carries an expression cassette forRep78 and an expression cassette for VP1 or VP2/VP3, while the secondvector carries an expression cassette for Rep52 and an expressioncassette for the remaining capsid protein(s) (VP2/VP3 if the firstvector expresses VP1, or VP1 if the first vector expresses VP2/VP3). Insome embodiments, the Rep78 cassette uses a promoter identical (orhaving similar strength) to the promoter in the Rep52 cassette, andoptionally has a suboptimal start codon, such that the Rep78 and Rep52proteins can be produced with an optimal stoichiometry. In otherembodiments, the Rep78 cassette uses a promoter weaker than the promoterin the Rep52 cassette, but has a canonical start codon, such that theRep78 and Rep52 proteins can be produced with an optimal stoichiometry.

In some embodiments, the VP1 cassette uses a promoter identical (orhaving similar strength) to the promoter in the VP2/VP3 cassette, andoptionally has a suboptimal start codon, such that the three capsidproteins can be produced with an optimal stoichiometry. In otherembodiments, the VP1 cassette uses a promoter weaker than the promoterin the VP2/VP3 cassette, but has a canonical start codon, such that thecapsid proteins can be produced with an optimal stoichiometry.

To reduce the number of baculoviral vectors needed to transduce therAAV-producing cells, the coding sequence for the rAAV genome may alsobe included in one of the two vectors, for example, in the second vector(i.e., the vector that carries the Rep52 expression cassette).

The present inventors have discovered that expressing Rep78 and Rep52from separate cassettes and expressing VP1 and VP2/VP3 from separatecassettes greatly improve the potency and/or yield of the rAAV producedin the insect cell systems. As demonstrated in the Working Examplebelow, the present systems produce rAAV with a multi-fold increase inpotency and/or yield as compared to production systems in which thecapsid proteins are produced from the prevailing one-vector system, inwhich the capsid proteins are produced from a single Cap gene and theRep proteins are produced from a single Rep gene.

The present production systems can be used to produce rAAV of anyserotype, such as AAV1, AAV2, AAV3, AAV3A, AAV3B, AAV4, AAV5, AAV6,AAV7, AAV8, AAV8.2, AAV9, AAVrh10, or an engineered serotype. The codingsequences of the capsid proteins may be derived from any desiredserotype.

The present systems also can be used to produce rAAV of a pseudotypesuch as AAV2/8, AAV2/5, or AAV2/6. By “pseudotype,” “pseudotyped,” or“cross-packaged” rAAV is meant a recombinant AAV whose capsid isreplaced with the capsid of another AAV serotype, to, for example, altertransduction efficacy or tropism profiles of the virus (see, e.g.,Balaji et al., J Surg Res. (2013) 184 (1):691-8). For example, an AAV2/8pseudotyped AAV contains capsids of AAV8 and the ITRs derived from AAV2.

The present systems also may be used to produce a chimeric or hybridAAV. By “chimeric” or “hybrid” rAAV is meant a recombinant AAV whosecapsid is assembled from capsid proteins derived from differentserotypes and/or whose capsid proteins are chimeric proteins withsequences derived from different serotypes (e.g., serotypes 1 and 2;see, e.g., Hauck et al., Mol Ther. (2003) 7 (3):419-25).

I. Capsid Protein Expression Cassettes

In mammalian cells, the Cap gene is transcribed into two mRNAs under thecontrol of the mammalian p40 promoter. One mRNA is translated to VP1,and the other into VP2 and VP3. The translation start codon for VP2 isACG, a suboptimal start codon that often causes ribosome skip, whereasthe start codon for VP3 is the canonical ATG. Through an interplay ofalternative splicing and the weak VP2 start codon, the Cap gene producesVP1, VP2, and VP3 in an apparent protein ratio of 1:1:10 (see, e.g.,Berns and Parrish, supra).

Failures of prior insect cell systems to produce rAAV with high yieldand potency have been attributed in part to the inability of thesesystems to achieve an optimal stoichiometry of the capsid proteins. Inthe present rAAV production systems, two separate expression cassettesare used for expressing VP1 and VP2/VP3. The use of separate expressioncassettes for VP1 and VP2/VP3 surprisingly leads to high yieldproduction of potent rAAV.

In some embodiments, the expression cassette for VP1 and the expressioncassette for VP2/VP3 use insect promoters of the same or similarstrengths. For example, the two cassettes may use identical promoters.Examples of insect promoters that can be used are a p10 promoter, a p35promoter, a polyhedron (polyh) promoter, an E1 promoter, a ΔE1 promoter,a 4×Hsp27 EcRE+minimal Hsp70 promoter, and a basic promoter.

The expression cassettes may contain additional regulatory elements,such as Kozak sequences; transcription initiation and termination sites(either modified or unmodified); mRNA splice sites (either modified orunmodified, within or adjacent to the polypeptide coding sequence; andviral, eukaryotic, or prokaryotic RNA elements that control splicing,nuclear export, localization, stabilization, or translation of mRNAs(e.g., Woodchuck hepatitis virus posttranscriptional regulatory element(WPRE) (Zufferey et al., J Virol. (1999) 73 (4):2886-92), MMLV/MPMV,eukaryotic constitutive transport element (CTE) (Li et al., Nature(2006) 443 (7108):234-7), RNA zipcodes (Jambhekar and DeRisi, RNA (2007)13 (5):625-42), and omega or other 5′-UTR RNA elements that increasetranslational efficiency).

In some embodiments, the coding sequences for the capsid proteins may bemodified to further enhance AAV yield and potency. For example, thecoding sequences may be codon-modified to increase mRNA stability,translation efficiency, and/or DNA vector stability in insect cells. Thestart codon regions of the coding sequences may also be modified, tofurther fine tune expression levels of the capsid proteins; for example,the VP1 start codon may be changed from wildtype ATG to a suboptimalcodon, such as the start codon of VP2 (ACG), such that VP1 expressionlevels are lower. The VP1 start codon may also be changed to othersuboptimal start codons such as TTG, CTG, and GTG.

To avoid production of VP2 and VP3 or any other by-product peptide fromthe VP1 expression cassettes, the VP1 coding sequence may be mutated toremove the native start codons for the embedded VP2 and VP3 ORFs, anyout-of-frame ATG sites, any undesired splice acceptor sites, any crypticpromoter sequences, and/or any destabilizing elements (see, e.g., Smithet al., supra).

In some embodiments, the present production systems produce rAAV6. Thecomplete amino acid sequence of an AAV6 VP1 protein is shown below,where the start site of VP2 (T) and the mutated start site of VP3(changed from the native M) are boldfaced and underlined:

(SEQ ID NO: 1)   1XAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD  61KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF QERLQEDTSF GGNLGRAVFQ 121AKKRVLEPFG LVEEGAK T AP GKKRPVEQSP QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE 181SVPDPQPLGE PPATPAAVGP TTV V ASGGGAE MADNNEGADG VGNASGNWHC DSTWLGDRVI 241TTSTRTWALP TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 301INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ LPYVLGSAHQ 361GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP SQMLRTGNNF TFSYTFEDVP 421FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ NQSGSAQNKD LLFSRGSPAG MSVQPKNWLP 481GPCYRQQRVS KTKTDNNNSN FTWTGASKYN LNGRESIINP GTAMASHKDD KDKFFPMSGV 541MIFGKESAGA SNTALDNVMI TDEEEIKATN PVATERFGTV AVNLQSSSTD PATGDVHVMG 601ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPPA 661EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VDFTVDNNGL 721YTEPRPIGTR YLTRPLIn the sequence above, X in position 1 may be M (wildtype; source:GenBank AAB95450.1), T, L, or V. In other embodiments, the VP1 startresidue may be another amino acid encoded by a non-canonical start codonsuch as a suboptimal start codon (see, e.g., Kearse et al., Genes Dev.(2017) 31:1717-31). In some embodiments, the mutated VP3 start site inthe VP1 protein is an amino acid other than the V shown above. In someembodiments, the AAV6 VP2 protein produced herein comprises a sequencespanning amino acids 138 to 736 of SEQ ID NO:1 where the VP3 start siteis not mutated (i.e., is the native methionine) and the AAV6 VP3 proteincomprises a sequence spanning amino acids 204-736 of SEQ ID NO:1 wherethe N-terminal amino acid is the native M.

Additional, non-limiting examples of coding sequence modifications aredescribed below. See also WO 2020/168145, the disclosure of which isincorporated hereby by reference in its entirety.

A. Modifications to Reduce Proteolytic Degradation of VP1 and VP2

In some embodiments, the VP1 and VP2 coding sequences may be mutated intwo or more codons such that the VP1 and VP2 proteins encoded therebyare resistant to proteolytic degradation during the production process.Because the VP1 coding sequence also contains the open reading frame forAAP, the codon changes may also cause mutations in the AAP. Thesemutations in VP1 and VP2 further improve the rAAV's infectivity, i.e.,potency.

In some embodiments, the present improved baculovirus-insect cellsystems can be used to produce any AAV serotype that is susceptible toproteolysis in insect cells. Such AAV serotypes may include, forexample, AAV1, AAV6, AAV8, or variants thereof, or any pseudotyped orchimeric rAAV whose VP1 and VP2 proteins are susceptible to proteolysisin insect cells.

In an improved AAV production system of the present disclosure, two ormore point mutations are introduced to AAV VP1 and VP2 proteins derivedfrom, e.g., AAV1, AAV4, AAV6, AAV7, AAV8, or AAV11, to remove the sitessusceptible to proteolysis in insect cells. The introduced pointmutations may be residues identical to those at the correspondingpositions in AAV2, AAV3, AAV5, AAV9, or AAV10.

By “corresponding” amino acid residue or region is meant an amino acidresidue or region that aligns with (though not necessarily identical to)the reference residue or region, when the subject sequence and thereference sequence containing the residues or regions are aligned toachieve maximum homology (allowing gaps that are recognized in the art).For example, amino acid residue 189 (L) of AAV8 VP1 corresponds to aminoacid residue 188 of AAV6 VP1.

In these systems, the overlapping region shared by the VP1 and VP2proteins may be mutated to remove proteolytic sites. For example, therAAV6 VP1 and/or VP2 may comprise mutations relative to the wildtype attwo or more residues in a region corresponding to residues 138-203(e.g., residues 151-201, residues 157-201, or residues 185-194 (i.e.,PQPLGEPPAT (SEQ ID NO:2), where cleavage has been shown between G andE)) of SEQ ID NO:1, where the mutated residues are selected from a groupconsisting of residues 157, 162, 164, 179, 188, 194, 196, 197, 200, and201. In some embodiments, the AAV6 VP1 and/or VP2 comprise two or moremutations selected from the group consisting of S157A, T162S, Q164A,S179T, L188I, T194A, A196S, A197G, P200S, and T201L (numbering accordingto SEQ ID NO:1). For example, an AAV6 VP1 and/or VP2 may have themutations (i) S179T, L1881, T194A, A196S, A197G, P200S, and T201L(“Variant 1”); (ii) S179T, L188I, T194A, A196S, and A197G (“Variant 2”);(iii) T194A, A196S, A197G, P200S, and T201L (“Variant 3”); (iv) S157A,T162S, and Q164A (“Variant 4”); or (v) P200S and T201L. For convenience,only the residue numbers in VP1 are referred to herein. The numbers ofthe corresponding residues in VP2 can be readily discerned from SEQ IDNO:1 above. For example, residue S157 in VP1 is residue S20 in VP2.

In particular embodiments, the AAV6 VP1 and/or VP2 comprise the mutationL188I and one or more other mutations in the group. For example, theAAV6 VP1 and/or VP2 may have the mutations L188I, P200S, and T201L.

In some embodiments, the AAV6 VP1 and/or VP2 comprise all ten mutationsof S157A, T162S, Q164A, S179T, L188I, T194A, A196S, A197G, P200S, andT201L, such that their sequence in the region corresponding to residues157-201 in SEQ ID NO:1 is as follows.

(SEQ ID NO: 4) AGIGKSGAQP AKKRLNFGQT GDTESVPDPQ PIGEPPAAPS GVGSLThis sequence is identical to the sequence in the corresponding regionin AAV9 VP1 and VP2.

Thus, to generate a modified VP1 coding sequence, one can replace thecoding sequence for residues 157-201 of VP1 (i.e., residues 20-64 ofVP2; SEQ ID NO:3) with the coding sequence for the corresponding aminoacid sequence (SEQ ID NO:4) of AAV9, using well known molecular clonetechniques; the resultant modified baculoviral AAV6 helper vector isreferred to herein as “AAV9 Transplant” and the resultant VP1 proteinsare referred to herein as “AAV6/9 VP1.” The same transplant may be madeto mutate the same region in VP2, resulting in an AAV6/9 VP2. In someembodiments, the portion of the AAV6 VP1 gene sequence that is replacedcomprises the underlined sequence shown below (see also FIG. 2 ):

(SEQ ID NO: 13) AAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTCCCCGACCCACAACCTCTCGGAGAACCTCCAGCAACCCCCGCTGCTGTGGGACCTACTACAATGGCTTCAGGCGGTGGCGCACCAATGG 

In certain embodiments, the underlined portion in SEQ ID NO: 13 isreplaced by a corresponding Cap gene sequence from AAV9 to generate anAAV9 Transplant disclosed herein. In particular embodiments, the AAV9Cap sequence transplanted to the AAV6 Cap gene in an AAV9 Transplantcomprises the underlined sequence shown below (see also FIG. 2 ):

(SEQ ID NO: 14) AAGAGCCAGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGCGGTGGCGCACCAATGG 

In other embodiments, a modified cap6 gene in the baculoviral AAV6helper construct can be generated by replacing the coding sequence forresidues 157-201 of VP1 in SEQ ID NO:1 (i.e., residues 20-64 of VP2; SEQID NO:3) with the coding sequence for the corresponding amino acidsequence from a serotype whose VP1 and/or VP2 are resistant toproteolytic cleavage in insect cells, e.g., AAV2, AAV3, AAV5, or AAV10.

In other embodiments, the VP1/VP2 proteins of AAV serotypes, such asAAV1, AAV4, AAV7, AAV8, or AAV11, can be mutated such that they containone or more mutations shown in FIG. 1C. The resultant proteins areexpected to be more resistant to proteolysis in insect cells. Unlimitingexamples of engineered VP1/VP2 proteins from various AAV serotypes areshown below:

(1) the engineered AAV1 VP1/VP2 proteins may contain one or more of thefollowing mutations: S158T/A, I160T/V, T163A/S/K, Q165K/A, K169R,S180A/T, L189E/I, T195A, A197S/T, A198S/G, V199L, P201T/S, and T202N/L;

(2) the engineered AAV4 VP1/VP2 proteins may contain one or more of thefollowing mutations: T158S/A, I160T/V, K163A/S, K165Q/A, K169R, K171R,V173N, E175G, D176Q/E, E177T, T178G, G179D/E, A180S/T, G181D/E, D182S,G183V, E189L/I, S191Q/E, T192P, S193P, G194A, M196P, S197T, D200G,D201T/S, S202N/L, and E203T;

(3) the engineered AAV6 VP1/VP2 proteins may contain one or more of thefollowing mutations: S158T/A, I160T/V, T163A/S/K, Q165K/A, K169R,S180A/T, L189E/I, T195A, A197S/T, A198S/G, V199L, P201T/S, and T202N/L;

(4) the engineered AAV7 VP1/VP2 proteins may contain one or more of thefollowing mutations: T158S/A, I160T/V, K163A/S, Q165K/A, R169K, S180A/T,L189E/I, S197T, S198G, V199L, and G202N/L;

(5) the engineered AAV8 VP1/VP2 proteins may contain one or more of thefollowing mutations: T158S/A, I160T/V, K163A/S, Q165K/A, R169K, S180A/T,L189E/I, S197T, G198S, V199L, P201T/S, and N202L; and

(6) the engineered AAV11 VP1/VP2 proteins may contain one or more of thefollowing mutations: S158T/A, I160T/V, K163A/S, K165Q/A, R169K, E175G,E176Q, D177T, T178G, G179D/E, A180S/T, G181D/E, D182S, G183V, E189L/I,S191Q/E, D192P, T193P, S194A, M196P, S197T, S200G, D201T/S, I202N/L, andE203T (consensus numbering; see FIG. 1C).

In some embodiments, the VP1/VP2 proteins from an AAV serotypesusceptible to proteolysis in insect cells are mutated to contain one ormore mutations at the following amino acid residues (consensus numberingin FIG. 1C): 158, 163, 165, 180, 189, 195, 197, 198, 201, and 202.(These residues correspond respectively to amino acid residues 157, 162,164, 179, 188, 194, 196, 197, 200, and 201 according to the numbering ofSEQ ID NO:1 (AAV6) or a corresponding sequence from another serotypealigned to show maximal homology to SEQ ID NO:1.) In furtherembodiments, the VP1/VP2 proteins may additionally contain one or moremutations at the following amino acid residues (consensus numbering inFIG. 1C): 160, 169, 171-179, 181-183, 191-194, 196, 199, and 203.

In some embodiments, the VP1/VP2 proteins from an AAV serotypesusceptible to proteolysis in insect cells are mutated to contain one ormore mutations: S158T/A, I160T/V, K/T163S/A/T, Q165K/A, K169R, K171R,V173N, E175G, D176Q/E, D/E177T, T178G, G179D/E, S180A/T, G181D/E, D182S,G183V, L189E/I, Q/S191E, T/D192P, S/T193P, G/S194A, T/G195A, M196P,A197S/T, A198G/S, V199L, S200G, D/P201S/T, T/S202N/L, and E203T(consensus numbering in FIG. 1C).

In some embodiments, the engineered VP1/VP2 proteins may contain one ormore of the following mutations: S158A, T163S, Q165A, S180T, L189I,T195A, A197S, A198G, P201S, and T202L (consensus number in FIG. 1C).(These residues correspond respectively to amino acid residues S157A,T162S, Q164A, S179T, L188I, T194A, A196S, A197G, P200S, and T201Laccording to the numbering of SEQ ID NO:1 (AAV6) or a correspondingsequence from another serotype aligned to show maximal homology to SEQID NO:1.)

The mutations described herein remove sites in the AAV capsid proteinssusceptible to proteolytic cleavage in insect cells. Thus, a helperconstruct containing the modified Cap gene will give rise to rAAVproducts of higher purity and uniformity, as well as improved capsidprotein.

It has been surprisingly found that the point mutations introducedherein to the AAV VP1/VP2 unique region (i.e., region common to VP1 andVP2 but absent in VP3) can also significantly improve the rAAV's potencythrough a mechanism that does not rely on solely on the prevention ofproteolytic cleavage.

B. Modifications to Further Improvement of rAAV Production Yield

Additionally or alternatively, the VP1 coding sequence in thebaculoviral helper construct is mutated in one or more codons in theregion coding for the VP1 PLA2 domain such that the engineered PLA2domain acquires higher enzymatic activity. It has been shown that higherPLA2 enzymatic activity may lead to a higher yield of rAAV in the insectcell production system.

The present disclosure provides a baculovirus-insect system in which theVP1 coding sequence is altered in the VP1 unique region (i.e., regionpresent in VP1 but not in VP2 or VP3) to improve the production yield ofthe rAAV in insect cells. The VP1 unique region (corresponding toresidues 1-137 of SEQ ID NO:1) contains the PLA2 domain, and themutations in the region may increase the enzymatic activity of the PLA2domain in the resultant VP1 protein.

In some embodiments, the baculoviral helper construct of the presentdisclosure provides helper functions for the production of rAAV6 orrAAV9 and the VP1 expression cassette on a helper construct includes amodified AAV6 or AAV9 Cap gene (Cap6 or Cap9, respectively) with amutated PLA2 domain. The mutated PLA2 domain may comprise mutationsrelative to the wildtype at one or more positions in a regioncorresponding to residues 1-137 (e.g., 52-97 or 67-92) of SEQ ID NO:1,where the positions are selected from a group consisting of residues 67,81, 84, 85, and 92 (numbering of SEQ ID NO:1). In some embodiments, theAAV6 VP1 protein comprises one or more mutations selected from the groupconsisting of A67E, Q81R, K84D, A85S, and R92K (numbering according toSEQ ID NO:1). In particular embodiments, the AAV6 VP1 protein comprisesall of the five mutations, such that its sequence in the regioncorresponding to residues 52-97 (SEQ ID NO:5) in SEQ ID NO:1 is asfollows.

(SEQ ID NO: 6) YLGPFNGLDK GEPVNEADAA ALEHDKAYDR QLDSGDNPYL KYNHADThis sequence is identical to the sequence in the corresponding regionin AAV2 VP1. Thus, to generate a modified cap6 gene in the baculoviralAAV6 helper construct, one can replace the coding sequence for residues52-97 or 67-92 of VP1 with the coding sequence for the correspondingamino acid sequence of AAV2, using well known molecular cloningtechniques.

It has been surprisingly found that the point mutations introducedherein to the AAV VP1 unique region can significantly improve the VP1PLA2 domain's enzymatic activity and also lead to significantimprovement (e.g., two or more fold, three or more fold, four or morefold, or five or more fold) in the yield of the rAAV produced in insectcells.

In particular embodiments, the engineered Cap gene encodes an AAV6 VP1protein containing both the aforementioned mutations in the PLA2 domainand the aforementioned mutations that remove the proteolytic sites. Oneexemplary modified AAV6 VP1 protein (“AAV6/2/9 VP1”) has the followingsequence (where V203 may be M203 or another amino acid instead):

(SEQ ID NO: 7)   1MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLD  61KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF QERLQEDTSF GGNLGRAVFQ 121AKKRVLEPFG LVEEGAK T AP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE 181SVPDPQPIGE PPAAPSGVGS LT V ASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI 241TTSTRTWALP TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 301INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ LPYVLGSAHQ 361GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP SQMLRTGNNF TFSYTFEDVP 421FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ NQSGSAQNKD LLFSRGSPAG MSVQPKNWLP 481GPCYRQQRVS KTKTDNNNSN FTWTGASKYN LNGRESIINP GTAMASHKDD KDKFFPMSGV 541MIFGKESAGA SNTALDNVMI TDEEEIKATN PVATERFGTV AVNLQSSSTD PATGDVHVMG 601ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPPA 661EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VDFTVDNNGL 721YTEPRPIGTR YLTRPL 

Other exemplary modified AAV6 VP1 protein has the same sequence as SEQID NO:7 except that the start residue in position 1 is not present, oris T, L, V or another amino acid encoded by a non-canonical start codonsuch as a suboptimal start codon. A baculoviral helper constructencoding one of these modified AAV6 VP1 proteins can be used to producerAAV6 or a pseudotyped or chimeric rAAV with improved potency and yieldin insect cells.

In some embodiments, the engineered Cap gene encodes an AAV9 VP1 proteincontaining an AAV2 PLA2 domain. The wildtype AAV9 VP1 sequence(UniProtKB-Q6JC40 (Q6JC40_9VIRU)) is shown below, where the five aminoacid residues differing from the corresponding ones in the AAV9/2 VP1 inthe PLA2 domain are indicated with boldface and boxes:

(SEQ ID NO: 11) 1MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY 

61

121 AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE181 SVPDPQPIGE PPAAPSGVGS LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI241 TTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR301 LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAH361 EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENV421 PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP481 GPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS541 LIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG601 ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT661 AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV721 YSEPRPIGTR YLTRNL 

The wildtype AAV9 and AAV2 sequences in this region (residues 52-97 ofSEQ ID NO:11) are shown below, where the five amino acid changesrelative to wildtype AAV9 after the AAV2 sequence transplant areboldfaced and boxed:

AAV9:  (SEQ ID NO: 12)YLGPGNGLDK GEPVNAADAA ALEHDKAYDQ QLKAGDNPYL KYNHAD AAV2: (SEQ ID NO: 6)

In some embodiments, M203 in SEQ ID NO:11 may be replaced by V203 oranother amino acid encoded by a noncanonical start codon.

C. Modifications to AAP

In some embodiments, the VP1 coding sequence also encodes an engineeredAAP that has improved abilities to stabilize capsid protein and tofacilitate capsid assembly. It has been shown that when residues 1-30 ofAAV6 AAP (“AAP6”) are changed to residues 1-30 AAV9 AAP (“AAP9”), thepotency of the rAAV produced with the engineered helper construct issignificantly increased. Residues 1-30 of AAP6 and AAP9 are shown below,where the six amino acid differences in wildtype AAP9 relative towildtype AAP6 are indicated by boldface and boxes:

AAV6 AAP: (SEQ ID NO: 8) MATQSQSPTH NLSENLQQPP LLWDLLQWLQ AAV9 AAP:(SEQ ID NO: 9)

The complete AAV6 AAP wild-type sequence is shown below:

(SEQ ID NO: 10)MATQSQSPTH NLSENLQQPP LLWDLLQWLQ AVAHQWQTIT KAPTEWVMPQ EIGIAIPHGWATESSPPAPE HGPCPPITTT STSKSPVLQR GPATTTTTSA TAPPGGILIS TDSTAISHHVTGSDSSTTIG DSGPRDSTSS SSTSKSRRSR RMMASRPSLI TLPARFKSSR TRSTSCRTSSALRTRAASLR SRRTCS

In some embodiments, the engineered AAP protein comprises one or moremutations at residues 8, 10, 12, 17, 21, and 22 (numbering based onconsensus sequence in FIG. 4A). In further embodiments, the engineeredAAP protein may comprise one or more of the following amino acidresidues: Q8, L10, Q12, P17, Q21, and V22. In some embodiments, theengineered AAP protein may comprise one or more of the followingmutations relative to the wildtype AAP sequence: P8Q, H10L, L12Q, Q17P,L21Q, and L22V. Unlimiting examples of engineered, improved AAP proteinsare shown below:

(1) engineered AAP1 (AAP of serotype 1) comprising one or more of thefollowing mutations: P8Q, I9T, H10L, L12Q, L16H, Q17P, L21Q, L22V, andL26I;

(2) engineered AAP2 (AAP of serotype 2) comprising one or more of thefollowing mutations: E2A, T5S, Y7S, L8Q, P10L, S11N, L12Q, D14E, S15N,H16L, Q17P, L21Q, E24D, I26L, and R27Q;

(3) engineered AAP4 (AAP of serotype 4) comprising one or more of thefollowing mutations: E2A, Q3T, A4Q, T5S, D6Q, P7S, L12Q, R13S, D14E,Q15N, L16H, P17Q, E18Q, C22V, L23W, M24D, T25L, V26I/L, R27Q, and C28W;

(4) engineered AAP6 (AAP of serotype 6) comprising one or more of thefollowing mutations: P8Q, H10L, L12Q, L16H, Q17P, L21Q, L22V, and L26I;

(5) engineered AAP7 (AAP of serotype 7) comprising one or more of thefollowing mutations: P8Q, L12Q, L16H, Q17P, L21Q, L22V, and L26I;

(6) engineered AAP8 (AAP of serotype 8) comprising one or more of thefollowing mutations: F7S, L12Q, L16H, Q17P, R19P, L21Q, and I26L;

(7) engineered AAP10 (AAP of serotype 10) comprising one or more of thefollowing mutations: S3T, P8Q, Q12L, L16H, Q17P, A19P, L21Q, and V26I/L;and

(8) engineered AAP11 (AAP of serotype 11) comprising one or more of thefollowing mutations: E2A, P3T, E4Q, T5S, D6Q, P7S, L12Q, R13S, D14E,Q15N, I16L/H, P17Q, A18Q, C22V, L23W, Q24D, T25L, L26I, K27Q, and C28W(see also FIG. 4B).

To generate engineered AAP protein, one can perform point mutations.Because the open reading frame encoding AAP is embedded in the Cap geneand is translated merely by a frameshift, one may also transplant fromanother serotype a portion of the Cap gene containing the codingsequence for an N terminal portion of AAP (e.g., residues 1-30, residues1-28, residues 2-30, residues 2-28, etc.). In some embodiments, an AAV9Cap gene portion coding for an AAV9 VP1/VP2 unique region (i.e., regioncommon to VP1 and VP2 but absent in VP3), for example, a cap9 sequencecomprising nucleotides 18-151 of SEQ ID NO:14 is substituted for thecorresponding region of cap6, to generate an engineered VP1 expressioncassette expressing an engineered AAV6 VP1 with a portion of AAV9 VP1(e.g., so as to remove the proteolytic site(s) as discussed above), aswell as an engineered AAP6 whose N-terminal 1-30 amino acid residues arenow identical to those of AAPS. In some embodiments, the AAP mutationsare generated by the same method as described above for the AAV9 Capgene transplant.

Additional examples of capsid proteins are described in the Examplesbelow.

II. Rep Protein Expression Cassettes

In mammalian cells, the Rep gene is transcribed into a Rep78-encodingRNA under the control of the mammalian p5 promoter and a Rep52-encodingRNA under the mammalian p19 promoter situated within the Rep78 ORF (see,e.g., Urabe et al., 2002, supra). The start codons for both Rep78 andRep52 are ATG. It has been found that overexpression of Rep78 relativeto Rep52 adversely impacts the yield of rAAV.

Failures of prior insect cell systems to produce rAAV with high yieldhave been attributed in part to the inability of these systems toachieve an optimal stoichiometry of the Rep proteins. In the presentrAAV production systems, two separate expression cassettes are used forexpressing Rep78 and Rep52. In some embodiments, the expression cassettefor Rep78 and the expression cassette for Rep52 use insect promoters ofthe same or similar strengths; but the use of a weaker start codon(e.g., CTG, TTG, GTG, and ACG) for Rep78 helps achieve a desiredRep78:Rep52 protein ratio and high AAV yield. In certain embodiments,the two cassettes may use identical promoters such as those listed abovefor the capsid expression cassettes. In other embodiments, the Rep78expression cassette has a canonical start codon (ATG) but uses a weakerpromoter than the Rep52 expression cassette to achieve the desiredstoichiometry.

In some embodiments, the Rep expression cassettes may contain additionalregulatory elements such as those described above for the capsid proteinexpression cassettes.

In some embodiments, the coding sequences for the Rep proteins may bemodified to further enhance AAV yield and potency. For example, thecoding sequences may be codon-modified to increase mRNA stability,translation efficiency, or DNA vector stability in insect cells. Toavoid production of Rep52 or any other by-product peptide from the Rep78expression cassette, the Rep78 coding sequence may be mutated to removethe start codon for the embedded Rep52 ORF, any off-frame ATG sites, anyundesired splice acceptor sites, any cryptic promoter sequences, and/orany destabilizing elements (see, e.g., Smith et al., supra). Conversely,the Rep52 coding sequence may be trimmed to remove any Rep78 codingsequence upstream of the Rep52 start codon.

The use of separate expression cassettes for Rep78 and Rep52, togetherwith the use of a suboptimal start codon for Rep78, surprisingly leadsto high yield production of potent rAAV.

III. Production of rAAV in Insect Cells

Production of rAAV in insect cells can be performed as describedpreviously. See, e.g., Urabe et al., 2002 and 2006, supra; Chen et al.,supra; Smith et al., supra; Mietzsch et al., supra; WO 2007/046703, WO2007/148971, WO 2009/104964, WO 2013/036118, and WO 2008/024998, all ofwhich are incorporated herein by reference in their entirety.

The insect cells in the production methods of the present disclosurecomprise the dual baculoviral helper vectors described herein. Theinsect cells may be, without limitation, a cultured cell line such asBTI-TN-5B1-4 derived from Trichoplusia ni (High Five™, Thermo FisherScientific, Carlsbad, Calif.), Sf9 cells or Sf21 cells (both of whichare derived from Spodoptera frugiperda), Sf9 or TN368 cells withmammalian-type glycan profiles (GlycoBac), or Sf-RVN cells(MilliporeSigma), Sf9-13F12 cells (Rhadovirus-free cells; Ma et al.,Virology (2019) 536:125-33).

The rAAV may comprise within its capsid an AAV vector containing atransgene of interest. The transgene may encode a reporter protein fordetection using biochemical (luciferase, SEAP) or imaging (GFP, Venus,dTomato) techniques. The transgene may encode a therapeutic protein,including, without limitation, a chimeric antigen receptor (CAR), aC-peptide or insulin, collagen VII, IGF-I, lipoprotein lipase,fibrinogen, prothrombin, Factor V, Factor VIII, Factor IX, Factor XI,Factor XII, Factor XIII, von Willebrand factor, prekallikrein, highmolecular weight kininogen (Fitzgerald factor), fibronectin,antithrombin III, heparin cofactor II, protein C, protein S, protein Z,protein Z-related protease inhibitor, plasminogen, alpha 2-antiplasmin,tissue plasminogen activator, urokinase, plasminogen activatorinhibitor-1, plasminogen activator inhibitor-2, or any enzyme fortreating a lysosome storage disease such as alpha-galactosidase A (fortreating Fabry disease). The transgene may encode a sequence-specificbinding protein (e.g., ZFP, TALE, TALEN, or dCas9). In some embodiments,the ZFP may be a ZFP transcription factor (e.g., a fusion protein inwhich a ZFP domain is fused to a transcription factor), a zinc fingernuclease (e.g., a fusion protein in which a ZFP domain is fused to anuclease), or a ZFP base editor (e.g., a fusion protein in which a ZFPdomain is fused to a nucleobase editor). The transgene may carry asequence that can be incorporated into a specific site in the hostgenome (donor) by homologous recombination or non-homologous endjoining. The transgene may encode an immunogenic protein for vaccination(e.g., a tumor antigen).

The AAV vector may comprise transcription regulatory elements (e.g.,promoter and enhancer) that can direct and regulate expression of thetransgene in human cells. The AAV vector may also comprise an AAVcomplete or partial inverted terminal repeat (ITR) on one or both endsof the transgene expression cassette. ITRs are required for packagingand viral integration into the host (human) genome. The AAV vector maybe single-stranded or self-complementary.

IV. Pharmaceutical Compositions of rAAV

The rAAV capsids produced by the present methods can be formulated witha pharmaceutically acceptable carrier as a pharmaceutical composition.Formulations include, without limitation, suspensions in liquid oremulsified liquids. Pharmaceutically acceptable carriers include, forexample, water, saline, dextrose, glycerol, sucrose, or the like, andcombinations thereof. In addition, the composition may contain auxiliarysubstances, such as, wetting or emulsifying agents, pH buffering agents,stabilizing agents, or other reagents that enhance the effectiveness ofthe rAAV pharmaceutical composition.

The rAAV pharmaceutical composition may be delivered in vivo byadministration to the patient, for example, by systemic administration(e.g., intravenous, intraperitoneal, intramuscular, subdermal,intrathecal, or intracranial infusion) or local injections.Alternatively, the rAAV can be delivered to cells ex vivo, such as cellsexplanted from a patient (e.g., lymphocytes, bone marrow aspirates, ortissue biopsy) or allogeneic cells (e.g., universal donor cells such asuniversal CAR T cells), followed by introduction of the treated cellsinto the patient, usually after selection for cells that haveincorporated the rAAV vector.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Exemplarymethods and materials are described below, although methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention. In case ofconflict, the present specification, including definitions, willcontrol. Generally, nomenclature used in connection with, and techniquesof, cell and tissue culture, molecular biology, virology, immunology,microbiology, genetics, analytical chemistry, synthetic organicchemistry, medicinal and pharmaceutical chemistry, and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art. Enzymatic reactions andpurification techniques are performed according to manufacturer'sspecifications, as commonly accomplished in the art or as describedherein. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.Throughout this specification and embodiments, the words “have” and“comprise,” or variations such as “has,” “having,” “comprises,” or“comprising,” will be understood to imply the inclusion of a statedinteger or group of integers but not the exclusion of any other integeror group of integers. All publications and other references mentionedherein are incorporated by reference in their entirety. Although anumber of documents are cited herein, this citation does not constitutean admission that any of these documents forms part of the commongeneral knowledge in the art.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLES Example 1: Production of AAV in Sf9 Insect Cells

Currently, helper systems used for Sf9 AAV manufacturing rely on leakyscanning, alternative splicing, and/or internal promoters to express allthe Rep and capsid proteins required for AAV production (Smith et al.,supra; Chen et al., supra). Since AAV capsids assemble based on thestoichiometry of the available capsid proteins (Berns and Parrish,supra), we hypothesized that exerting greater control over the amount ofVP1 expressed during rAAV production could increase the amount of VP1incorporated into rAAV capsids and thereby the viral potency.

To test this hypothesis, we cloned the coding sequences for individualcapsid proteins into separate baculoviral vectors under the control ofindependent promoters. To allow greater control over the Rep proteins,we also cloned the coding sequences for individual Rep proteins intoseparate baculoviral vectors. In order to test various possiblecombinations of Rep and Cap coding sequences on different vectors, weconstructed two versions of the baculoviral helper systems (FIG. 5 ).

In the first version, we paired the Rep78 expression cassette with theVP1 expression cassette and paired the Rep52 expression cassette withthe VP2/VP3 expression cassette. In the second version, we paired theRep52 expression cassette with the VP1 expression cassette and pairedthe Rep78 expression cassette with the VP2/VP3 expression cassette. BothRep expression cassettes were under the control of a polyhedrinpromoter. Both the VP1 and VP2/3 expression cassettes were under thecontrol of a p10 promoter. With the exception of the VP2/3 gene, whichutilizes the same leaky scanning mechanism as in the natural context ofAAV replication, none of the Rep coding sequences and the VP1 codingsequence in these helper vectors relies on leaky scanning or alternativesplicing for expression. In order to reduce the number of baculoviralvectors required for rAAV production, we incorporated the codingsequence for the AAV minigenome into the Rep52-expressing vector in bothversions (FIG. 5 ). For the present study, the AAV minigenome containedthe expression cassette for alpha-galactosidase A (GLA) or zinc fingerproteins (ZFP) (SBS #65918 and SBS #57890, whose coding sequences wereseparated by a nucleotide linker encoding a T2A self-cleaving peptide inthe transgene expression cassette; see WO 2018/102665 and WO2020/072677). A GLA-encoding rAAV may be useful in gene therapy forFabry disease. The ZFP bound to and repressed the expression of themouse microtubule-associated protein tau (Mapt) gene.

The coding sequences in the expression cassettes and the encodedpolypeptide sequences are as follows. In the VP1 coding sequence, theATG start codon for the native VP3 ORF was changed to GTG, resulting inan M-to-V substitution. In the Rep78 coding sequence, the ATG startcodon for the native Rep58 ORF was changed to GTG, also resulting in anM-to-V substitution. For the studies herein, the Rep gene was derivedfrom AAV2.

Generation of rAAV and Determination of Relative Yield

Naïve Sf9 insect cells were inoculated with baculovirus-infected insectcells (BIIC1 and BIIC2 for cells respectively infected by the twobaculoviral vectors). The ratio of BIIC1:BIIC2:naïve Sf9 cells for theinoculation was 1:1:10000. Cell cultures were incubated for six days.The cells were then harvested, resuspended and freeze/thawed threetimes. The cell lysate was treated with benzonase. Cell debris wasremoved by centrifugation. AAV was concentrated by the addition of PEGand NaCl followed by incubation on ice and centrifugation ofprecipitated virus. Concentrated virus was resuspended and applied to aCsCl gradient followed by overnight ultracentrifugation. Banded AAV wascollected and dialyzed. Then qPCR was performed to titer the vectorgenome (vg) content. rAAV6 (with a GLA-encoding transgene) and rAAV9(with a ZFP-encoding transgene) tested herein were obtained by thesemethods. Total AAV yield was calculated by multiplying the vg/mL by thetotal mL of the sample and expressed relative to the yield of thestandard helper system 1, which expresses the AAV2 replicases and AAV6capsid proteins and employs the one-vector system described in Smith etal., supra.

Additional rAAV serotypes, rAAV1, rAAV2, and rAAV3B, were obtained bythe Version 2 method illustrated in FIG. 5 . The VP1 in the rAAV1 andrAAV3B viruses contained an AAV2 PLA2 domain as described above. In thecase of rAAV1, the VP1 and VP2 additionally contained an AAV9 transplantas described above to remove proteolytic sites. AAV2 and AAV3B did notappear susceptible to the proteolytic cleavage as observed with AAV1 andAAV6; and thus, their VP1/VP2 were not engineered to contain atransplanted AAV9 VP sequence). These three viruses also carried thesame ZFP transgene as the rAAV9.

In the studies, the AAV genomes included ITR (inverted terminal repeats)from AAV2 and the insect cells expressed AAV2 replicases.

The capsid protein sequences of the rAAV6, rAAV9, rAAV1, rAAV2, andrAAV3B viruses herein are shown below. The capsid proteins of rAAV6contained sequences from AAV2 and AAV9 as described in detail above. Thecapsid proteins of rAAV9, rAAV1, and rAAV3B also were engineered tocontain sequences from other AAV serotypes, as indicated below.

Determination of rAAV6 Potency

rAAV6 obtained by the method described above in this Example was appliedto HepG2 cells at a multiplicity of infection (MOI or virus/cell basedon the vg titer) of 900K. The cells were incubated for five days. Thetissue culture supernatant was collected and assayed for GLA enzymeactivity by measuring the level of a fluorescent reporter4-methylumbelliferyl (4-MU) released from a GLA substrate. Enzymaticactivity was calculated relative to activity with an rAAV sampleproduced by standard helper system 1.

Determination of rAAV9 Potency

rAAV9 obtained by the method described above in this Example was appliedto mouse cortical neurons at an MOI of 30K and incubated for six days.The cells were harvested for total RNA and used to generate cDNA. ThecDNA was analyzed by qPCR for expression of mouse Mapt mRNA and zincfinger protein (ZFP) mRNA encoded by the AAV transgene. Afternormalizing the mRNA expression to the host cell RNA, potency wascalculated relative to the rAAV9 produced by the AAV9 standard helpersystem (infra). The potency of the rAAV1, rAAV2, and rAAV3B viruses canbe tested in the same manner as well.

Sequences of Rep and Capsid Proteins

The amino acid and coding sequences for the rAAV6, rAAV1, rAAV2, rAAV3B,and rAAV9 viruses tested herein are shown below.

rAAV6 VP1 coding sequence: (SEQ ID NO: 15)ACGGCTGCCGACGGTTATCTACCCGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTCAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGAGGGTTCTCGAACCTTTTGGTCTGGTTGAGGAAGGTGCTAAGACCGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAGTGGCTTCAGGCGGTGGCGCACCAGTGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACATGGGCCTTGCCCACCTATAACAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTGCCATTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGTCACGACCATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCAGTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTGGGACGGTCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGATGCTGAGAACGGGCAATAACTTTACCTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCAGTACCTGTATTACCTGAACAGAACTCAGAATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCTGTTTAGCCGGGGGTCTCCAGCTGGCATGTCTGTTCAGCCCAAAAACTGGCTACCTGGACCCTGTTACCGGCAGCAGCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAACTTTACCTGGACTGGTGCTTCAAAATATAACCTTAATGGGCGTGAATCTATAATCAACCCTGGCACTGCTATGGCCTCACACAAAGACGACAAAGACAAGTTCTTTCCCATGAGCGGTGTCATGATTTTTGGAAAGGAGAGCGCCGGAGCTTCAAACACTGCATTGGACAATGTCATGATCACAGACGAAGAGGAAATCAAAGCCACTAACCCCGTGGCCACCGAAAGATTTGGGACTGTGGCAGTCAATCTCCAGAGCAGCAGCACAGACCCTGCGACCGGAGATGTGCATGTTATGGGAGCCTTACCTGGAATGGTGTGGCAAGACAGAGACGTATATCTGCAGGGTCCTATTTGGGCCAAAATTCCTCACACGGATGGACACTTTCACCCGTCTCCTCTCATGGGCGGCTTTGGACTTAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACGCCTGTTCCTGCGAATCCTCCGGCAGAGTTTTCGGCTACAAAGTTTGCTTCATTCATCACCCAGTATTCCACAGGACAAGTGAGCGTGGAGATTGAATGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCCGAAGTGCAGTATACATCTAACTATGCAAAATCTGCCAACGTTGATTTCACTGTGGACAACAATGGACTTTATACTGAGCCTCGCCCCATTGGCACCCGTTACCTCACCC GTCCCCTGTAA rAAV6 VP1 polypeptide: (SEQ ID NO: 16; X = M or T)XAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY KYLGPFNGLDKGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF QERLQEDTSF GGNLGRAVFQAKKRVLEPFG LVEEGAKTAP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTESVPDPQPIGE PPAAPSGVGS LTVASGGGAP VADNNEGADG VGNASGNWHC DSTWLGDRVITTSTRTWALP TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRLINNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ LPYVLGSAHQGCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP SQMLRTGNNF TFSYTFEDVPFHSSYAHSQS LDRLMNPLID QYLYYLNRTQ NQSGSAQNKD LLFSRGSPAG MSVQPKNWLPGPCYRQQRVS KTKTDNNNSN FTWTGASKYN LNGRESIINP GTAMASHKDD KDKFFPMSGVMIFGKESAGA SNTALDNVMI TDEEEIKATN PVATERFGTV AVNLQSSSTD PATGDVHVMGALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK NTPVPANPPAEFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VDFTVDNNGLYTEPRPIGTR YLTRPL rAAV6 VP2/VP3 coding sequence: (SEQ ID NO: 17)ACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCACAAGAGCCAGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGCGGTGGCGCACCAGTGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGCCTCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACATGGGCCTTGCCCACCTATAACAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTGCCATTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGGTCACGACGAATGATGGCGTCACGACCATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCAGTTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAGTACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTGGGACGGTCATCCTTTTACTGCCTGGAATATTTCCCATCGCAGATGCTGAGAACGGGCAATAACTTTACCTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCAGTACCTGTATTACCTGAACAGAACTCAGAATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCTGTTTAGCCGGGGGTCTCCAGCTGGCATGTCTGTTCAGCCCAAAAACTGGCTACCTGGACCCTGTTACCGGCAGCAGCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAACTTTACCTGGACTGGTGCTTCAAAATATAACCTTAATGGGCGTGAATCTATAATCAACCCTGGCACTGCTATGGCCTCACACAAAGACGACAAAGACAAGTTCTTTCCCATGAGCGGTGTCATGATTTTTGGAAAGGAGAGCGCCGGAGCTTCAAACACTGCATTGGACAATGTCATGATCACAGACGAAGAGGAAATCAAAGCCACTAACCCCGTGGCCACCGAAAGATTTGGGACTGTGGCAGTCAATCTCCAGAGCAGCAGCACAGACCCTGCGACCGGAGATGTGCATGTTATGGGAGCCTTACCTGGAATGGTGTGGCAAGACAGAGACGTATATCTGCAGGGTCCTATTTGGGCCAAAATTCCTCACACGGATGGACACTTTCACCCGTCTCCTCTCATGGGCGGCTTTGGACTTAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACGCCTGTTCCTGCGAATCCTCCGGCAGAGTTTTCGGCTACAAAGTTTGCTTCATTCATCACCCAGTATTCCACAGGACAAGTGAGCGTGGAGATTGAATGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCCGAAGTGCAGTATACATCTAACTATGCAAAATCTGCCAACGTTGATTTCACTGTGGACAACAATGGACTTTATACTGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGTCCCCTGTAArAAV6 VP2 polypeptide sequence: (SEQ ID NO: 18; X = M or T)XAPGKKRPVE QSPQEPDSSA GIGKSGAQPA KKRLNFGQTG DTESVPDPQP IGEPPAAPSGVGSLTMASGG GAPVADNNEG ADGVGNASGN WHCDSTWLGD RVITTSTRTW ALPTYNNHLYKQISSASTGA SNDNHYFGYS TPWGYFDFNR FHCHFSPRDW QRLINNNWGF RPKRLNFKLFNIQVKEVTTN DGVTTIANNL TSTVQVFSDS EYQLPYVLGS AHQGCLPPFP ADVFMIPQYGYLTLNNGSQA VGRSSFYCLE YFPSQMLRTG NNFTFSYTFE DVPFHSSYAH SQSLDRLMNPLIDQYLYYLN RTQNQSGSAQ NKDLLFSRGS PAGMSVQPKN WLPGPCYRQQ RVSKTKTDNNNSNFTWTGAS KYNLNGRESI INPGTAMASH KDDKDKFFPM SGVMIFGKES AGASNTALDNVMITDEEEIK ATNPVATERF GTVAVNLQSS STDPATGDVH VMGALPGMVW QDRDVYLQGPIWAKIPHTDG HFHPSPLMGG FGLKHPPPQI LIKNTPVPAN PPAEFSATKF ASFITQYSTGQVSVEIEWEL QKENSKRWNP EVQYTSNYAK SANVDFTVDN NGLYTEPRPI GTRYLTRPLrAAV6 VP3 polypeptide sequence: (SEQ ID NO: 19)MASGGGAPVA DNNEGADGVG NASGNWHCDS TWLGDRVITT STRTWALPTY NNHLYKQISSASTGASNDNH YFGYSTPWGY FDFNRFHCHF SPRDWQRLIN NNWGFRPKRL NFKLFNIQVKEVTTNDGVTT IANNLTSTVQ VFSDSEYQLP YVLGSAHQGC LPPFPADVFM IPQYGYLTLNNGSQAVGRSS FYCLEYFPSQ MLRTGNNFTF SYTFEDVPFH SSYAHSQSLD RLMNPLIDQYLYYLNRTQNQ SGSAQNKDLL FSRGSPAGMS VQPKNWLPGP CYRQQRVSKT KTDNNNSNFTWTGASKYNLN GRESIINPGT AMASHKDDKD KFFPMSGVMI FGKESAGASN TALDNVMITDEEEIKATNPV ATERFGTVAV NLQSSSTDPA TGDVHVMGAL PGMVWQDRDV YLQGPIWAKIPHTDGHFHPS PLMGGFGLKH PPPQILIKNT PVPANPPAEF SATKFASFIT QYSTGQVSVEIEWELQKENS KRWNPEVQYT SNYAKSANVD FTVDNNGLYT EPRPIGTRYL TRPLrAAV1 VP1/VP2/VP3 polypeptide sequences (transplanted AAV2 PLA2 domainis shown in box; transplanted AAV9 VP2/VP3 proteolytic resistant domainis italicized and boldfaced; the first amino acids of VP2 and VP3 (T138and V203, respectively, in SEQ ID NO:24) are boldfaced and underlined):

(SEQ ID NO: 24)

AKKRVLEPLG LVEEGAK T AP GKKRPVEQSP QEPDSS

 

 

 

 

T V ASGGGAP VADNNEGADG VGNASGNWHC DSTWLGDRVITTSTRTWALP TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRLINNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ LPYVLGSAHQGCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP SQMLRTGNNF TFSYTFEEVPFHSSYAHSQS LDRLMNPLID QYLYYLNRTQ NQSGSAQNKD LLFSRGSPAG MSVQPKNWLPGPCYRQQRVS KTKTDNNNSN FTWTGASKYN LNGRESIINP GTAMASHKDD EDKFFPMSGVMIFGKESAGA SNTALDNVMI TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMGALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK NTPVPANPPAEFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ YTSNYAKSAN VDFTVDNNGLYTEPRPIGTR YLTRPL rAAV2 VP1/VP2/VP3 polypeptide sequences (the first amino acids of VP2and VP3 (T138 and V203, respectively, in SEQ ID NO:25) are boldfaced andunderlined):

(SEQ ID NO: 25) TAADGYLPDW LEDTLSEGIR QWWKLKPGPPPPKPAERHKD DSRGLVLPGY KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNPYLKYNHADAEF QERLKEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEPVK T AP GKKRPVEHSPVEPDSSSGTG KAGQQPARKR LNFGQTGDAD SVPDPQPLGQ PPAAPSGLGT NT V ATGSGAPVADNNEGADG VGNSSGNWHC DSTWLGDRVI TTSTRTWALP TYNNHLYKQI SSQSGASNDNHYFGYSTPWG YFDFNRFHCH FSPRDWQRLI NNNWGFRPKR LNFKLFNIQV KEVTQNDGTTTIANNLTSTV QVFTDSEYQL PYVLGSAHQG CLPPFPADVF MVPQYGYLTL NNGSQAVGRSSFYCLEYFPS QMLRTGNNFT FSYTFEDVPF HSSYAHSQSL DRLMNPLIDQ YLYYLSRTNTPSGTTTQSRL QFSQAGASDI RDQSRNWLPG PCYRQQRVSK TSADNNNSEY SWTGATKYHLNGRDSLVNPG PAMASHKDDE EKFFPQSGVL IFGKQGSEKT NVDIEKVMIT DEEEIRTTNPVATEQYGSVS TNLQRGNRQA ATADVNTQGV LPGMVWQDRD VYLQGPIWAK IPHTDGHFHPSPLMGGFGLK HPPPQILIKN TPVPANPSTT FSAAKFASFI TQYSTGQVSV EIEWELQKENSKRWNPEIQY TSNYNKSVNV DFTVDTNGVY SEPRPIGTRY LTRNLrAAV3B VP1/VP2/VP3 polypeptide sequences (transplanted AAV2 PLA2 domainis shown in box; the first amino acids of VP2 and VP3 (T138 and V203,respectively, in SEQ ID NO:26) are boldfaced and underlined):

(SEC ID NO: 26)

AKKRILEPLG LVEEAAK T AP GKKRPVDQSP QEPDSSSGVG KSGKQPARKR LNFGQTGDSESVPDPQPLGE PPAAPTSLGS NT V ASGGGAP VADNNEGADG VGNSSGNWHC DSQWLGDRVITTSTRTWALP TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLINNNWGFRPKK LSFKLFNIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL PYVLGSAHQGCLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS QMLRTGNNFQ FSYTFEDVPFHSSYAHSQSL DRLMNPLIDQ YLYYLNRTQG TTSGTTNQSR LLFSQAGPQS MSLQARNWLPGPCYRQQRLS KTANDNNNSN FPWTAASKYH LNGRDSLVNP GPAMASHKDD EEKFFPMHGNLIFGKEGTTA SNAELDNVMI TDEEEIRTTN PVATEQYGTV ANNLQSSNTA PTTRTVNDQGALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK NTPVPANPPTTFSPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYNKSVN VDFTVDTNGVYSEPRPIGTR YLTRNL rAAV9 VP1/VP2/VP3 polypeptide sequences (transplanted AAV2 PLA2 domainis shown in box; the first amino acids of VP2 and VP3 (T138 and V203,respectively, in SEQ ID NO:27) are boldfaced and underlined):

(SEC ID NO: 27)

AKKRLLEPLG LVEEAAK T AP GKKRPVEQSP QEPDSSAGIG KSGAQPAKKR LNFGQTGDTESVPDPQPIGE PPAAPSGVGS LT V ASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVITTSTRTWALP TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQRLINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY QLPYVLGSAHEGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF PSQMLRTGNN FQFSYEFENVPFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT INGSGQNQQT LKFSVAGPSN MAVQGRNYIPGPSYRQQRVS TTVTQNNNSE FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGSLIFGKQGTGR DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQGILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPTAFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGVYSEPRPIGTR YLTRNL AAV2 Rep78 coding sequence:

(SEQ ID NO: 20) CTGGCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAGTGGGAGTTGCCGCCAGATTCTGACTTGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAGTGGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACTTACACGTGCTCGTGGAAACCACCGGGGTGAAATCCTTAGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAACGGCGCCGGAGGCGGGAACAAGGTGGTGGACGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATTTAGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGACGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACGTGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTG CATCTTTGAACAATAAAAV2 Rep78 polypeptide:

(SEQ ID NO: 21; X = M or L)XAGFYEIVIK VPSDLDEHLP GISDSFVNWV AEKEWELPPD SDLDLNLIEQ APLTVAEKLQRDFLTEWRRV SKAPEALFFV QFEKGESYFH LHVLVETTGV KSLVLGRFLS QIREKLIQRIYRGIEPTLPN WFAVTKTRNG AGGGNKVVDE CYIPNYLLPK TQPELQWAWT NLEQYLSACLNLTERKRLVA QHLTHVSQTQ EQNKENQNPN SDAPVIRSKT SARYVELVGW LVDKGITSEKQWIQEDQASY ISFNAASNSR SQIKAALDNA GKIMSLTKTA PDYLVGQQPV EDISSNRIYKILELNGYDPQ YAASVFLGWA TKKFGKRNTI WLFGPATTGK TNIAEAIAHT VPFYGCVNWTNENFPFNDCV DKMVIWWEEG KMTAKVVESA KAILGGSKVR VDQKCKSSAQ IDPTPVIVTSNTNMCAVIDG NSTTFEHQQP LQDRMFKFEL TRRLDHDFGK VTKQEVKDFF RWAKDHVVEVEHEFYVKKGG AKKRPAPSDA DISEPKRVRE SVAQPSTSDA EASINYADRY QNKCSRHVGMNLMLFPCRQC ERMNQNSNIC FTHGQKDCLE CFPVSESQPV SVVKKAYQKL CYIHHIMGKVPDACTACDLV NVDLDDCIFE QAAV2 Rep52 coding sequence:

(SEQ ID NO: 22) ATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAAAV2 Rep52 polypeptide:

(SEQ ID NO: 23)MELVGWLVDK GITSEKQWIQ EDQASYISFN AASNSRSQIK AALDNAGKIM SLTKTAPDYLVGQQPVEDIS SNRIYKILEL NGYDPQYAAS VFLGWATKKF GKRNTIWLFG PATTGKTNIAEAIAHTVPFY GCVNWTNENF PFNDCVDKMV IWWEEGKMTA KVVESAKAIL GGSKVRVDQKCKSSAQIDPT PVIVTSNTNM CAVIDGNSTT FEHQQPLQDR MFKFELTRRL DHDFGKVTKQEVKDFFRWAK DHVVEVEHEF YVKKGGAKKR PAPSDADISE PKRVRESVAQ PSTSDAEASINYADRYQNKC SRHVGMNLML FPCRQCERMN QNSNICFTHG QKDCLECFPV SESQPVSVVKKAYQKLCYIH HIMGKVPDAC TACDLVNVDL DDCIFEQ

Results

We tested the ability of each version in FIG. 5 to produce rAAV6 and thepotency of the resulting rAAV with respect to standard helper system 1and standard helper system 2, which differs from standard helper system1 in that its capsid expression cassettes use the AAV6/2/9 hybridsequences described above. Surprisingly, the pairing of the Rep and Capgenes had a significant impact on both yield and potency. Version 1 hadan approximately two-fold reduction in yield and eight-fold increase inpotency, whereas Version 2 had an approximately four-fold increase inyield and five-fold increase in potency (Table 1). Comparison of thecapsid ratios showed that the Version 1 rAAV6 had very little VP2 and aVP1:VP3 ratio similar to that observed with wildtype AAV (1:10).Recombinant AAV produced by the Version 2 system had a similar capsidratio to that observed with standard helper system 2 (Table 1).

TABLE 1 Characterization of rAAV6* Relative Yield Potency Capsid ratioHelper (N = 3) (N = 3) VP1 VP2 VP3 Standard helper system 1 1.0 1.0 1 266 Standard helper system 2 3.8 3.1 1 1.6 24 Version 1 0.5 8.5 1 0.00514 Version 2 4.1 5.2 1 1.4 26 *The yield and potency numbers areaverages of three independent production lots. The capsid ratios arefrom a single production lot.

For rAAV9, Version 1 had an approximately three-fold reduction in yieldand similar potency, whereas Version 2 had an approximately four-foldincrease in yield and two-fold decrease in potency, as compared tostandard helper system 1 (Table 2). Comparison of the capsid ratiosshowed that the Version 1 rAAV9 had half as much VP2 as the AAV9standard helper system and a VP1:VP3 ratio one half of that observedwith wildtype AAV (1:10). Recombinant AAV9 produced by the Version 2system had a similar capsid ratio to that observed with rAAV9 providedby the AAV9 standard helper system (Table 2). The AAV9 standard helpersystem was the same as the AAV6 standard helper system 2, except thatinstead of producing AAV6/2/9 hybrid capsid proteins, it produced hybridAAV9 capsid proteins in which the VP1 protein was engineered to containan AAV2 PLA2 domain as described above.

TABLE 2 Characterization of rAAV9* Relative Yield Potency Capsid ratioHelper (N = 6) (N = 6) VP1 VP2 VP3 AAV9 standard helper 1.0 1.0 1 1.7 18Version 1 0.3 0.9 1 0.8 20 Version 2 4.0 0.5 1 1.5 26 *The yield andpotency numbers and capsid ratios are from six dependent productionlots.

Additional serotypes rAAV1, rAAV2 and rAAV3B were successfully producedby the Version 2 method of FIG. 5 .

These data suggest that the pairing of Rep and Cap expression cassetteson a helper vector influences the yield and potency of produced rAAV.Furthermore, the data demonstrate that the Rep and Cap proteins can besuccessfully expressed from two separate baculoviral vectors and helpproduce infectious rAAV at high yield. Lastly, these data demonstratethat the production system described herein is broadly applicable todifferent AAV serotypes and is not restricted to the more extensivelytested AAV6 and AAV9 versions.

TABLE 3 List of Sequences SEQ ID NO Description 1 AAV6 VP1 amino acidsequence 2 AAV6 proteolytic site 3 AAV6 VP1/VP2 fragment 4 AAV9 VP1/VP2fragment 5 AAV6 VP1 fragment 6 AAV2 VP1 fragment 7 AAV6/2/9 VP1 aminoacid sequence 8 AAP6 residues 1-30 9 AAP9 residues 1-30 10 AAP6 aminoacid sequence 11 AAV9 VP1 amino acid sequence 12 AAV9 VP1 fragment 13Partial AAV6 Cap nucleotide sequence 14 Partial AAV9 Cap nucleotidesequence 15 VP1 coding sequence of rAAV6 in Example 1 16 VP1 polypeptidesequence of rAAV6 in Example 1 17 VP2/VP3 coding sequence of rAAV6 inExample 1 18 VP2 polypeptide sequence of rAAV6 in Example 1 19 VP3polypeptide sequence of rAAV6 in Example 1 20 AAV2 Rep78 coding sequencein Example 1 21 AAV2 Rep78 polypeptide sequence in Example 1 22 AAV2Rep52 coding sequence in Example 1 23 AAV2 Rep52 polypeptide sequence inExample 1 24 VP1/VP2/VP3 polypeptide sequences of rAAV1 in Example 1 25VP1/VP2/VP3 polypeptide sequences of rAAV2 in Example 1 26 VP1/VP2/VP3polypeptide sequences of rAAV3B in Example 1 27 VP1/VP2/VP3 polypeptidesequences of rAAV9 in Example 1 28-73 Sequences in figures

What is claimed is:
 1. An insect cell comprising a first baculoviralvector comprising an expression cassette for Rep78 and a secondbaculoviral vector comprising an expression cassette for Rep52, whereinthe first vector and the second vector further comprise (i) anexpression cassette for VP1 and an expression cassette for VP2/VP3,respectively; or (ii) an expression cassette for VP2/VP3 and anexpression cassette for VP1, respectively.
 2. The insect cell of claim1, wherein the Rep78 expression cassette and the Rep52 expressioncassette comprise identical insect promoters.
 3. The insect cells ofclaim 1 or 2, wherein the Rep78 expression cassette comprises anon-canonical start codon for the Rep78 coding sequence, wherein thecodon is optionally ACG, TTG, GTG, or CTG.
 4. The insect cell of any oneof claims 1-3, wherein the VP1 expression cassette and the VP2/VP3expression cassette comprise identical insect promoters.
 5. The insectcell of any one of claims 1-4, wherein the VP1, VP2, and VP3 proteinscomprise amino acid sequences from the same AAV serotype, or from morethan one AAV serotype.
 6. The insect cell of claim 5, wherein the VP1,VP2, and/or VP3 proteins comprise amino acid sequences from AAV1, AAV2,AAV3 (optionally AAV3B), AAV6, and/or AAV9.
 7. The insect cell of anyone of claims 1-6, wherein the Rep78 and Rep52 proteins are derived froma different AAV serotype from the VP1, VP2, and/or VP3 proteins.
 8. Theinsect cell of any one of claims 1-7, wherein (i) the VP1 comprises SEQID NO:1, 7, or 16 with or without the first amino acid residue;  the VP2comprises amino acid residues 138-736 or 139-736 of SEQ ID NO:1 or 7, orcomprises SEQ ID NO:18 with or without the first amino acid residue; and the VP3 comprises amino acid residues of 204-736 or 205-736 of SEQ IDNO:1 or amino acid residues 203-736 or 204-736 of SEQ ID NO:7, orcomprises SEQ ID NO:19 with or without the first amino acid residue; or(ii) the VP1 comprises SEQ ID NO:24 with or without the first amino acidresidue;  the VP2 comprises amino acid residues 138-736 or 139-736 ofSEQ ID NO:24; and  the VP3 comprises amino acid residues of 203-736 or204-736 of SEQ ID NO:24; or (iii) the VP1 comprises SEQ ID NO:25 with orwithout the first amino acid residue;  the VP2 comprises amino acidresidues 138-735 or 139-735 of SEQ ID NO:25; and  the VP3 comprises203-735 or 204-735 of SEQ ID NO:25; or (iv) the VP1 comprises SEQ IDNO:26 with or without the first amino acid residue;  the VP2 comprisesamino acid residues 138-736 or 139-736 of SEQ ID NO:26; and  the VP3comprises amino acid residues of 203-736 or 204-736 of SEQ ID NO:26; or(v) the VP1 comprises SEQ ID NO:27 with or without the first amino acidresidue;  the VP2 comprises amino acid residues 138-736 or 139-736 ofSEQ ID NO:27; and  the VP3 comprises amino acid residues of 203-736 or204-736 of SEQ ID NO:27; and optionally wherein the Rep78 comprises SEQID NO:21 with or without the first amino acid residue; and/or the Rep52comprises SEQ ID NO:23 with or without the first amino acid residue. 9.The insect cell of any one of claims 1-8, wherein the Rep78, Rep52, VP1,and VP2/VP3 expression cassettes each comprise an insect promoterselected from a polyhedron promoter, an IE-1 promoter, and a p10promoter.
 10. The insect cell of any one of claims 1-9, wherein thefirst baculoviral vector comprises an expression cassette for VP2/VP3,and the second baculoviral vector comprises an expression cassette forVP1.
 11. The insect cell of any one of claims 1-10, wherein one or bothof the first and second vectors are stably integrated into the genome ofthe insect cell.
 12. The insect cell of any one of claims 1-11, whereinthe cell is an Sf9 or Sf21 cell.
 13. The insect cell of any one ofclaims 1-12, further comprising a coding sequence for a recombinant AAVgenome, wherein the recombinant AAV genome comprises an expressioncassette for a transgene of interest that is under the transcriptionalcontrol of a mammalian promoter, and an AAV inverted terminal repeat(ITR) on both termini.
 14. The insect cell of claim 13, wherein thecoding sequence for the recombinant AAV genome is located on the firstor second vector, or is located on a third vector.
 15. The insect cellof claim 13 or 14, wherein the transgene of interest encodes atherapeutic protein, optionally selected from a zinc finger protein(ZFP) transcription factor and a protein whose function is lacking ordeficient in a genetic disease.
 16. The insect cell of claim 13 or 14,wherein the transgene of interest encodes a gene editing protein,optionally selected from a zinc finger nuclease, a ZFP deaminase, a ZFPrecombinase, a TALEN, a CRISPR Cas protein, and a CRISPR Cpf protein.17. A method of producing a recombinant AAV virion, comprising:providing an insect cell of any one of claims 13-16, culturing theinsect cell under conditions that allow expression of the recombinantAAV genome and packaging of the recombinant AAV genome within an AAVcapsid comprising the VP1, VP2, and VP3 proteins, and isolating theproduced recombinant AAV virion from the culture.
 18. A recombinant AAVvirion produced in the insect cell of any one of claims 13-16 or by themethod of claim
 17. 19. A pharmaceutical composition comprising therecombinant AAV virion of claim 18 and a pharmaceutically acceptablecarrier.
 20. A combination of expression cassettes in any one of claims1-16.